Reducing sodium in soy sauce fermentation while preserving flavor and safety presents significant technical challenges. To address this, we constructed a synthetic microbial community (SynMC) comprising Tetragenococcus halophilus T10, Zygosaccharomyces rouxii QH-25, and Wickerhamiella versatilis CGMCC 3790 for reduced-salt fermentation (13 %NaCl). Multi-omics analysis revealed three coordinated improvements: First, microbial community stabilization through suppression of spoilage taxa (e.g., Millerozyma) and enrichment of functional genera (e.g., Wickerhamiella), reducing negative ecological correlations from 5.3 % to 1.7 % compared to the low-salinity control group. Second, metabolic restructuring enhanced characteristic aromas while reducing biogenic amines (BAs) to 21.98 mg/L (76 % lower than L group). Third, metatranscriptomics identified upregulated amino acid metabolism (238 % more BA-degrading enzymes) and carbohydrate utilization pathways. These synergistic effects indicate that strategic microbial consortia design can overcome the salt reduction challenge through targeted ecological and metabolic regulation, enabling industrial-scale production of superior, safer low-salt soy sauce.

Aquaculture constitutes a significant reservoir for antibiotic-resistant bacteria (ARB) posing substantial risks to seafood consumers and public health. This study aimed to reveal the most contaminated stages and spots of foodborne pathogens, and ARB along the whole fish production chain in Greece. To reach this aim, samples (water, surfaces, fish feed, and fish) were collected from multiple spots (e.g. surfaces, equipment) of all stages of the entire chain, from farming to packaging, and analyzed for their microbiological and antibiotic resistance status through classical and molecular approaches. At population point of view, surfaces from broodstock, larvae and pre-fattening tanks were found to be the most burdened spots in farming stages, recording levels close to 5 log cfu/ml or g or cm, while the most burdened spots were found to be on working surfaces before and after decontamination, the concrete floor, transport boxes for fish and fish samples in processing. Based on High-Resolution Melting analysis (HRM) followed by Sanger sequencing, the potential pathogenic microbiota, mainly consisting of Enterococcus spp., Serratia spp., Morganella morganii and Klebsiella pneumoniae. at both pre-fattening and processing stages, indicating the possible transfer of these microorganisms along the fish production chain. These potential pathogens, mainly isolated from surfaces and water of weaning and pre-fattening tanks, fish feeds, working surfaces, fish transport boxes, concrete floors and fish especially from transport boxes and harvesting, presented a noteworthy resistance to most antibiotics tested (e.g., Clindamycin, Nalidixic acid, Streptomycin, Sulphonamides, Ceftazidime, Erythromycin, Vancomycin, Penicillin G, and Cefoxitin). The findings underscore the importance of early detection and intervention strategies to mitigate ARB transmission, thereby enhancing the safety and quality of aquaculture products.

This study evaluated the survival of Salmonella enterica and Escherichia coli O157:H7 on watercress (Nasturtium officinale R.Br), mint (Mentha spicata L.), and basil (Ocimum basilicum L.) during refrigerated storage. The efficacy of non-thermal technologies in inactivating these pathogens on basil leaves was investigated, using dielectric barrier discharge (DBD) cold plasma (15 min at 50, 500, and 1000 Hz), glow discharge plasma (15 min at gas flows of 10, 20, and 30 mL/min; and 20 or 30 min at gas flow 20 mL/min), and pulsed UV-light (PL) at doses of 758, 1,516, and 2280 mJ/cm (all below FDA-maximum PL dose of 12 J/cm). Morphological alterations in bacterial cells following DBD and PL treatments were examined using scanning electron microscopy. Basil showed significantly higher survival of S. enterica and E. coli O157:H7 during storage (p < 0.05). DBD 500 Hz treatment caused the higher reduction (∼2.30 log CFU/g) of both pathogens, compared to 50 and 1000 Hz. Glow discharge plasma was more effective at a gas flow rate of 20 mL/min compared to 10 and 30 mL/min after 15 min (p < 0.05) for either pathogen. Treatment with glow discharge plasma at 20 mL/min for 20 min achieved reductions of >5.0 log CFU/g for E. coli O157:H7 and 3.46 log CFU/g for S. enterica. The highest PL dose tested resulted in reductions of 3.60 and 3.10 log CFU/g for E. coli O157:H7 and S. enterica, respectively. These findings highlight the potential of glow discharge plasma, particularly for targeting E. coli O157:H7 on basil leaves.

In March 2024, highly pathogenic avian influenza (HPAIV) H5N1 was first detected in U.S. dairy cattle and has since spread to herds across at least 17 states. Infected cows typically present with mastitis, decreased milk production, and poor milk quality with high viral loads in milk. While commercial pasteurization of milk effectively inactivates avian influenza virus (AIV), growing consumer interest in raw milk and derived products raises public health concerns due to the risk of zoonotic transmission. Standard yogurt production includes an initial heating step at 82 °C for 30 min to denature milk proteins which also inactivates AIV. However, some home yogurt recipes omit this initial heating step. This project determined whether AIV present in raw milk could remain viable through fermentation and persist in the final yogurt product. Raw milk (ca. pH 6.7) was spiked with AIV (ca. 6.6 log 50 % egg infectious doses (EID) per mL and inoculated with a commercial starter culture to produce yogurt. The viability of the virus was determined before and after fermentation (ca. 7.3 h) at 42 °C with resultant pH drop ≤4.4. A significant (p < 0.05) reduction of viable AIV (≥4.1 log EID) was observed in both the yogurt and the control samples of raw milk incubated at 42 °C but without starter culture (ca. pH 6.63). Viral inactivation was likely due to a combination of incubation at a sublethal temperature, pH below 4.4, and microbial degradation. Thus, properly fermented yogurt has a negligible risk of transmitting AIV to humans.

C. perfringens, an anaerobic bacterium, is a common cause of food poisoning that can persist on surfaces in slaughterhouses. However, the mechanisms governing its survival in such environments - characterised by variations in relative air humidity (RAH) - remain poorly understood. This study aimed to evaluate the effect of air exposure on C. perfringens survival and to identify the mechanisms responsible for its inactivation. Vegetative cells and spores of C. perfringens were deposited on inert surfaces and exposed to different RAH (11 %, 43 %, 75 %, 100 %) under both aerobic and anaerobic conditions, to assess the contributions of osmotic and oxidative effects induced by dehydration to cell death. At low RAH, more than 99 % of vegetative cells were inactivated within one day, regardless of oxygen presence. Epifluorescence microscopy and flow cytometry analyses revealed that dehydration and rehydration disrupted membrane integrity, contributing to inactivation through lethal mechanical damage. At 100 % RAH, vegetative cells survived over 3 days under aerobic conditions (>1 %) and over 30 days under anaerobic conditions (>0.003 %). The composition of the dehydration medium had little effect on cell survival. In contrast, spores were much more resistant, with around 10 % survival after two months of stress in presence of oxygen, without any significant effect of dehydration. These results highlight the potential of exploiting RAH fluctuations to develop control strategies targeting C. perfringens vegetative cells. However, the extreme resilience of spores confirms the need for specific and targeted decontamination methods to eliminate them effectively.

Fermented red peppers (FRPs) provide distinct flavor and possible health benefits, but understanding of their microbial functions, viral diversity, pathogenicity, and horizontal gene transfer (HGT) patterns remains limited. Integrated multi-method analysis revealed FRP's bacterial community was dominated by Bacillus (21.52 %), Lactobacillus sensu lato (14.27 %), and Pantoea (13.60 %). Bacillus drove core fermentation with an over 40 % contribution to carbon degradation and iron reduction. The virome was dominated by Caudoviricetes phages, yet 25.5 % of the functions of viral genes remained unknown. Critically, multidrug resistance genes were the most abundant ARGs, and beneficial bacteria served as major reservoirs for ARGs, co-occurring with potential opportunistic pathogens. Despite inhibitory conditions, these last dominated key metabolic nodes hydrogen generation and acetate oxidation. Counterintuitively, ARG profiles correlated with bacterial composition but not with mobile genetic elements or detected HGT events, challenging HGT as the primary ARG driver. These findings necessitate dual strategies: leveraging key microbes for fermentation efficiency while implementing stringent monitoring to mitigate pathogen and ARG related risks.

For wine, higher alcohols are double-edged swords, which benefit flavors in appropriate amount but cause negative effects on aromas as well as side-effects for consumers in excessive amount. In this study, five kinds of ammonium salts were tested to reduce higher alcohols produced by Zygosaccharomyces mellis LGL-1 (LGL-1), among which, (NH)HPO exhibited most excellent effects with higher alcohols reduced by 32.79 %. Both NH and HPO demonstrated individual capacities to reduce higher alcohol levels and transcriptome analysis was employed to investigate the synergistic mechanism. Results showed that both NH and HPO could regulate higher alcohol biosynthesis, but with different regulators, Cat8 for the synergistic effect of NH and HPO, and Ino80 for HPO specifically. Meanwhile, HPO reduced the activity of BCAT2, specifically and NH increased the activity of BCAT1 particularly, the combination of which led to the reduction of higher alcohol biosynthesis. Both Gtr1 and TorC1 were downregulated with HPO or NH addition, with more pronounced downregulations under their combination. Collectively, the synergistic effects of ammonium and phosphorus on higher alcohol biosynthesis in LGL-1 were existed. To amplify this effect, the molar ratio of nitrogen to phosphorus was optimized and a mole ratio of 1:1 was applied for wampee/grape winemaking with higher alcohols reduced by 24.99 %-26.76 % but with the content of ethanol, esters and organic acid unchanged significantly. Data above suggested that the combined addition of NH and HPO could play synergistic effects on higher alcohol biosynthesis, which was an effective strategy to reduce higher alcohols in wines for improved qualities.

This study investigated the antiviral potential of exopolysaccharides (EPSs) from probiotic bacteria against human noroviruses (hNoVs). EPSs from Bacillus subtilis CU1, B. subtilis R0179, and Lactiplantibacillus plantarum 299V were initially evaluated using Tulane virus (TV), a cultivable hNoV surrogate. Although EPSs reduced the cytopathic effects caused by TV infection, no clear dose-response relationship was observed. In contrast, the zebrafish model enabled testing of hNoV strains and revealed a distinct anti-hNoV GII.4 activity specific to EPS from B. subtilis CU1. This effect was virus genotype- and bacteria strain-specific: EPSs from B. subtilis R0179 and L. plantarum 299V showed no activity, nor did CU1 EPS affect hNoV GII.2 or GII.17. The major EPS fraction was identified as a levan composed of β-(2,6)-linked Fruf, which exhibited high binding affinity to hNoV GII.4 virus-like particles and P particles, confirmed by saliva-binding ELISA and bio-layer interferometry. Finally, B. subtilis CU1 was used to ferment carrot juice. The antiviral effect of EPS produced by B. subtilis CU1 in fermented carrot juice was validated, and the EPS yield was optimized accordingly. These findings highlight B. subtilis CU1 EPS as a promising anti-hNoVs agent and demonstrate carrot juice as a safe, cost-effective substrate for scalable production of functional probiotic EPSs.

Food losses and waste have become a major worldwide challenge, partly due to mold spoilage at the consumer level. One possible way to reduce food waste due to moldy foods would be to avoid a too conservative approach where products are directly discarded if fungal growth is observed. However, a food safety risk exists as many fungal species produce potentially toxic mycotoxins that can migrate into foods, so this hazard needs to be considered to establish consumer recommendations. This study quantified citrinin and ochratoxin A accumulation and migration in strawberry jams after inoculation with Penicillium verrucosum UBOCC 109221 and incubation at 8 and 20 °C. The mold failed to grow after 28days of incubation at 20 °C on jam with 59% sugar content but exhibited a constant growth rate of about 1.15 mm/d on the other sugar concentrations. After 14 days of incubation, citrinin concentration (10000 ng/g) for the jam containing 34 % sugar was about twice the concentration observed for 39 and 44 % sugar. Mycotoxin migration experiments were then carried out for 39 % sugar content and showed that the maximum mycotoxin concentrations were obtained for the 5 cm lesion diameter. At 20 °C, citrinin concentration (30 000 ng/g) was about twice that obtained at 8 °C, while the maximum ochratoxin A concentration was about 100 ng/g. For 1 and 2 cm lesions with 8°C storage, mycotoxins were not detected at 3 cm depth, accordingly jam can be consumed after removing about 2 to 3 cm beyond the moldy area. For greater lesions, jam should be discarded because mycotoxins were detected at >4 cm depth.

Foodborne viral infections are a global public health concern, yet quantitative risk assessments (QMRA) for enteric viruses in bivalve mollusks are scarce, especially in South America. We conducted Argentina's first QMRA for five human enteric viruses- norovirus (NoV), rotavirus A (RVA), hepatitis A (HAV), hepatitis E (HEV), and adenovirus (AdV) in bivalve mollusks. From August 2018-March 2023, 390 specimens from Golfo Nuevo, Chubut, were pooled (n = 113) and analyzed by real time PCR. A national survey revealed 3.6 % of servings were raw or minimally cooked. AdV showed the highest prevalence and viral load, followed by NoV GII and RVA; HAV and HEV had lower loads, and NoV GI was undetected. Per-serving infection risks peaked for AdV and NoV GII from raw oyster and mussel consumption. Individual annual infection probabilities were 2.44 × 10 for AdV, 1.33 × 10 for NoV GII, and 6.60 × 10 for HAV. RVA and HEV posed lower risks. Sensitivity analysis identified AdV and HAV prevalence, and mussel and clam consumption, as key drivers of the shellfish-associated infection risk. Our findings pinpoint raw and undercooked oysters and mussels as significant sources of risk. This QMRA provides crucial, country-specific evidence to support optimizing disinfection processes and enhancing monitoring of critical environmental contamination sources along the seafood production and distribution chain.

Sichuan Shai vinegar (SSV) is a traditional fermented product with complex microbial ecology. This study elucidated the functional division of labor within the yeast microbiota during the early stage (days 1-5) of solid-state fermentation in SSV. Investigating of four key yeast strains (Saccharomyces cerevisiae, Pichia kudriavzevii, Kazachstania humilis, and Brettanomyces bruxellensis) via co-culturing, metabolomics, and simulated fermentation revealed distinct roles: Pichia kudriavzevii dominated ester synthesis, Brettanomyces bruxellensis primarily produced characteristic flavor compounds (e.g., acetaldehyde, 4-ethylguaiacol), Kazachstania humilis efficiently produced acids accelerating acidification, and Saccharomyces cerevisiae produced ethanol, which served as a precursor for ester synthesis by other yeasts. The triple-strain combination of Pichia kudriavzevii, Kazachstania humilis, and Brettanomyces bruxellensis exhibited optimal synergy, achieving peak total acid (10.96 g/100 g DW) and acetic acid (3.54 g/100 g DW) content while significantly enhancing characteristic flavor profiles. Untargeted metabolomics indicated that this combination efficiently regulated multiple flavor biosynthesis pathways through pyruvate-mediated metabolic hubs. This systematic clarification of functional roles within the yeast community provides an experimental foundation for designing synthetic microbial starters to modulate flavor profiles and advance the standardization of fermented food production.

Our previous research suggested that the co-cultivation of Escherichia coli and Saccharomyces cerevisiae could enhance the oxidative tolerance of yeast, and identified hexadecanoic acid as a key metabolic regulator. Nevertheless, the precise regulatory mechanisms remain unclear. The objective of this work is to elucidate the mechanism through which hexadecanoic acid enhances the oxidative tolerance of S. cerevisiae. Results suggested that addition of hexadecanoic acid could significantly reduce the reactive oxygen species (ROS) level by 25.78 % and contribute to maintaining the structural integrity of S. cerevisiae. Hexadecanoic acid reduced ROS production through inhibiting the activation of the Ca signaling pathway. On the other hand, due to the decreased generation of intracellular ROS, hexadecanoic acid also down-regulated the antioxidant defense capability of S. cerevisiae, suggesting the non-necessity of excessive activation of the antioxidant system. Transcriptomic analysis revealed hexadecanoic acid treatment associated 27 up-regulated and 30 down-regulated genes. Based on the transcriptomics analysis results and previous findings, the MF(α)2 was chosen as a target gene to be clarified. Further construction of the MF(α)2 knockout strain demonstrated that the oxidative tolerance of ΔMF(α)2 strain significantly decreased. In light of these findings, it can be inferred that hexadecanoic acid may promote the MF(α)2 gene expression, thereby delaying the cell cycle and enhancing yeast oxidative tolerance. Results presented in this work would contribute to the understanding of the regulatory mechanisms of hexadecanoic acid on the oxidative tolerance of S. cerevisiae, and would also offer insights into the potential for manual intervention to regulate the oxidative tolerance of strains.

This study pioneered an integrated investigation of superheated steam (SHS) sterilization by quantifying kinetics and unraveling dual-action mechanisms against foodborne pathogens (Salmonella Typhimurium, Listeria monocytogenes and Staphylococcus aureus) on pork belly surfaces. Sterilization kinetics of SHS with different treatment temperature (160-200 °C) and flow rates (20-30 kg h) during 60 s were modeled using Weibull and Logistic equations. Comparatively, the Logistic equation was rigorously validated as superior (R ≥ 0.998, RMSE ≤ 0.097, Af ≤ 1.183), enabling precise prediction of microbial inactivation dynamics. Kinetic analysis revealed a novel biphasic pattern: rapid pathogen reduction (≤20 s) followed by a distinct tailing phase (20-60 s), challenging conventional single-phase sterilization assumptions. Mechanistically, SHS induced immediate disruption of cell wall/membrane integrity, evidenced by a decline from 2.07 to 2.25 to 0.52-0.75 King units·(100 mL) in AKP activity, an increase from 2.88 to 2.98 to 3.93-4.18 mS cm in conductivity, and concurrent surges in nucleic acid/protein leakage within 20 s. Critically, ATPase activity plummeted 68-77 %, from 3.46 to 3.53 to 0.8-1.1 U·mg prot, directly linking membrane destabilization to energy metabolism collapse. These findings established SHS as a multi-modal intervention, synergizing thermal inactivation with targeted biochemical disruption of microbial homeostasis (cellular ion balance disruption, material exchange, and ATPase activity interference). By providing mechanistic insights and predictive modeling tools, this research validated SHS as a scalable, eco-friendly alternative to chemical sanitizers, reducing antimicrobial resistance risks and environmental footprint in meat processing.

Fungi and mycotoxins hugely contaminate food and feed commodities and consequently contribute to adverse effects on health and the economy. While various measures have been adopted to control food contamination, the potential application of nanotechnology to control fungi and mycotoxins in food seems promising; nevertheless, it has not been sufficiently explored. This study investigated the antifungal effect of plant-mediated silver nanoparticles (AgNPs) form Garcinia kola, Carica papaya, Achillea millefolium, Ocimum gratissimum, and Perilla frutescens against ten food-borne fungi. Furthermore, the effect of Garcinia kola AgNPs and Achillea millefolium AgNPs against aflatoxin (AFB1 and AFB2) and ochratoxin (OTA and OTB) production by Aspergillus flavus, Aspergillus ochraceus, and Aspergillus clavatus was determined by using an Ultra Performance Liquid Chromatography-Triple Quadrupole Spectrometry (UPLC-MS/MS) instrument to quantify the amount of aflatoxins and ochratoxins produced following AgNPs antifungal treatments. The characterization analysis of AgNPs showed that stable and crystalline AgNPs were bioformulated, with sizes ranging from 10.9 to 67.5 nm. The antifungal effect of AgNPs by agar well diffusion method after 7 days of incubation revealed that at a concentration of 100 mg/L, AgNPs were able to exhibit an antifungal effect on the tested food-borne pathogens. As found, a maximum inhibitory zone (MIZ) of 20.3 mm by Penicillium frutescens AgNPs on Penicillium chrysogenum was obtained, while a 10.7 mm MIZ exhibited by Carica papaya AgNPs on Aspergillus Niger was recorded. Mycelial growth inhibition (MGI) activity of AgNPs at varying concentrations of 6.25, 12.5, 25, 50, and 100 mg/L was established. The highest MGI of AgNPs of 100 % was recorded against Penicillium citrinum, while the lowest MGI of 6.7 % for Aspergillus flavus was noted. The significant difference at p ≤ 0.05 was validated by comparing MIZ and MGI induced by the tested AgNPs to the control groups. The ability of AgNPs derived from Garcinia kola and Achillea millefolium showed a reduction in mycotoxin (AFB, AFB, OTA, and OTB) production as the concentration of the synthesized AgNPs increased. These findings demonstrate that AgNPs have immense potential as antifungal agents for controlling the growth of fungi and the subsequent biosynthesis of their respective mycotoxins.

Foodborne pathogens remain a significant public health concern, with fresh and minimally processed vegetables serving as potential transmission vectors. Acanthamoeba castellanii, a free-living protozoan, can harbor pathogenic bacteria within its cysts, potentially enhancing their survival under adverse conditions. While pickling is known to reduce microbial contamination through acidic and osmotic stress, its efficacy against pathogens protected within Acanthamoeba cysts remains unclear. This study investigated the survival of Salmonella Typhimurium, Escherichia coli O157:H7, Listeria monocytogenes, and Staphylococcus aureus within A. castellanii cysts on fresh and pickled cucumbers and cauliflowers over storage periods of 9 and 21 days, respectively. Coculture experiments revealed that E. coli O157:H7 was internalized by A. castellanii trophozoites at significantly higher rates than the other pathogens. Encystment rates increased over time but were unaffected by the bacterial species harbored within the amoebae. On fresh produce, although bacterial release into nutrient-rich medium during excystment varied by pathogen and produce type, Acanthamoeba cysts consistently served as a protective reservoir, maintaining viable intracellular populations throughout the storage period. In pickled samples, E. coli O157:H7 and L. monocytogenes exhibited prolonged survival, releasing into nutrient-rich medium during excystment for up to 16 days and persisting within trophozoites. S. aureus showed extended retention inside excysted trophozoites, while S. Typhimurium displayed lower but consistent survival. These findings highlight the protective role of A. castellanii cysts, which enable foodborne pathogens to withstand the acidic and osmotic stresses encountered during pickling. The study underscores the potential risk of protozoan-mediated pathogen persistence in both fresh and pickled produce, emphasizing the need for improved food safety interventions that account for amoeba-bacteria interactions.

Brettanomyces bruxellensis is a significant spoilage yeast in wine, known for producing volatile phenols that alter wine aroma and quality. These compounds are formed from hydroxycinnamic acids (HCA), including ferulic, p-coumaric, and caffeic acids, which are naturally present in grapes and in wine. HCA conversion to ethyl phenols is an original and conserved characteristic of B. bruxellensis that results from the presence of a two-step pathway. We hypothesized HCA conversion serves as a detoxification strategy for B. bruxellensis. This study investigated how different HCA concentrations affect growth and viability of 24 B. bruxellensis strains, with a particular focus on two wine-isolated strains (L1760 and 2OT13_02). Under wine-like conditions, only p-coumaric acid exhibited a cytotoxic effect, affecting growth, cell viability, and the conversion of HCA to vinylphenol (VP) and ethylphenol (EP) in a concentration-dependent manner. This effect was specifically induced by p-coumaric acid itself, rather than by its VP or EP derivatives. It was more pronounced at pH 3.3, likely due to enhanced diffusion of the acidic form across yeast membrane. The two strains showed a similar behavior, although with a different degree of tolerance. Sensitivity of the 24-strain panel to HCA showed a large diversity, possibly linked to genetic traits. Our findings highlight the potential of using naturally occurring wine compounds like p-coumaric acid as part of an integrated strategy to control B. bruxellensis population in wine.

Cronobacter spp. is an opportunistic foodborne pathogen responsible for life-threatening infections such as meningitis and necrotizing enterocolitis in neonates, but also for various complications in elderly and immunocompromised people. Environmental monitoring in powdered milk manufacturing plants shows that Cronobacter can persist in production environments for long periods of time. The aim of this study is to develop a method to assess the viability of Cronobacter cells after implementation of stress encountered in industries to determine whether this bacterium can persist in processing environments in a dormant state known as Viable But Non-Cultivable (VBNC) state, making it undetectable by conventional enumeration methods. We developed two detection systems specific to the genus Cronobacter based on quantitative PCR (qPCR) and Droplet Digital PCR (ddPCR) in combination with a PMAxx™ (Propidium Monoazide) treatment and agar enumeration to assess the viability of detected cells. Despite a better sensitivity for ddPCR, qPCR-PMAxx™ was more suitable for VBNC detection, as it effectively differentiates viable from dead cells. Desiccation at 58 °C and 20 % relative humidity led to a significant reduction in culturable bacteria, with a drop from 6.82 to 2.58 log copies of genome per coupon over 48 h, while qPCR-PMAxx™ revealed that the proportion of VBNC cells represents approximately 1/100 of the total population of viable bacteria (V). Similarly, treatment of Cronobacter biofilms with a peracetic acid containing disinfectant induced the VBNC state in Cronobacter. The ability of this bacterium to enter the VBNC state may explain its long-term survival in processing plants and highlights the need for appropriate detection methods for effective environmental monitoring plans.

Listeria monocytogenes, the leading cause of fatalities worldwide among foodborne pathogens, poses serious risks to food safety and public health. Therefore, a rapid and accurate detection method is crucial for early interception and effective management. In this study, a one-pot LAMP-CRISPR/Cas12b detection system based on the lmo0753 gene was developed for rapid detection of L. monocytogenes by combining loop-mediated isothermal amplification (LAMP) with a CRISPR/Cas12b assay. Further integration of a lateral flow assay (LFA) to develop a LAMP-CRISPR/Cas12b-LFA assay enabled direct detection of the results on the strips with the naked eye. Nine L. monocytogenes strains belonging to eight serotypes tested positive with both the one-pot LAMP-CRISPR/Cas12b and LAMP-CRISPR/Cas12b-LFA assays. Two assays did not show cross-reactivity with L. innocua and eight other foodborne bacteria. The limits of detection were 10 CFU/mL for pure culture and 20 CFU/g for spiked pork samples. Moreover, the enrichment time was substantially shortened to 3 h for pork samples spiked with only L. monocytogenes F2365, and 4-5 h for pork samples spiked with mixed bacteria. In addition, with one-pot LAMP-CRISPR/Cas12b detection, 5 of 66 fresh pork samples, 1 of 20 ready-to-eat food samples, and 2 of 24 raw milk samples tested positive for L. monocytogenes, in agreement with the results obtained through a culture based standard method. Thus, this study established one-pot LAMP-CRISPR/Cas12b and LAMP-CRISPR/Cas12b-LFA assays for rapid, visual detection of L. monocytogenes in food samples.

Superheated steam (SHS) is an emerging sanitation technology for dry food processing environments. This study investigates the efficacy of antimicrobial pretreatments in conjunction with SHS to inactivate microorganisms on stainless steel surfaces. Stainless steel coupons were inoculated with Enterococcus faecium (11.1 ± 0.4 log CFU/cm), equilibrated to a water activity of 0.2 (a), and subjected to various antimicrobial pretreatments prior to SHS exposure. This includes soaking for 10 or 20 s in sterile water, 70 % ethanol, 0.05 % peracetic acid (mist applied for 5 s), or acidified oil emulsion (100 μL). SHS experiments were conducted using both bench-scale (100 % steam content) and pilot-scale equipment (5 % steam, 95 % hot air). The nozzle-to-surface distance was maintained at 3 cm. All experiments were performed in triplicate, and surviving microorganisms were enumerated using plate counts (detection limit: 0.2 log CFU/cm). Antimicrobial pretreatments alone resulted in microbial reductions of <2.0 ± 0.5 log CFU/cm. SHS treatment using the bench-scale system achieved >6.4 ± 0.3 log CFU/cm reduction regardless of pretreatment. Pilot-scale SHS treatment (with or without antimicrobials) achieved lower reductions (<2.8 ± 0.2 log CFU/cm, p < 0.05) possibly due to lower steam content. Increasing the treatment intensity (195 °C for 30 s) resulted moderate improvement in microbial inactivation (3.3 ± 0.6 log CFU/cm). This study demonstrates that antimicrobial pretreatments can enhance the efficacy of SHS sanitation and highlights the importance of steam content in the treatment environment.

Hongmeiren citrus, an economically important fruit, currently lacks targeted measures to control postharvest decay. While durian shells are rich in diverse antimicrobial compounds, their antifungal potential remains underexplored. In this study, Fusarium oxysporum was identified as a dominant pathogen causing rot in Hongmeiren citrus. We then evaluated the antifungal efficacy of durian shell water extract (DSWE) against F. oxysporum in vitro and further elucidated the underlying inhibitory mechanism through molecular docking analysis. Our results demonstrated that treatment with 4 mg/mL DSWE significantly reduced mycelial growth diameter by 77.2 % and completely suppressed sporulation by the 6th day post-inoculation, compared to the control (CK). Propidium iodide (PI) staining and scanning electron microscopy (SEM) revealed severe membrane damage and hyphal collapse in F. oxysporum treated with 4 mg/mL DSWE. Molecular docking predicted that three bioactive compounds in DSWE (fraxetin, taxifolin, and muscone) could bind to key proteins involved in the cell wall and membrane biosynthesis of F. oxysporum. Experimental validation confirmed that fraxetin, taxifolin, and muscone reduced the colony diameter of F. oxysporum by 62.8 %, 53.2 %, and 21.1 %, respectively, while inducing significant membrane injury. Notably, fraxetin and taxifolin exhibited superior antifungal activity compared to muscone. This study elucidates the mechanism by which DSWE inhibits F. oxysporum and highlights its potential as an eco-friendly strategy for controlling postharvest pathogens in citrus.